Chronic
Inhibition Reduces Intimal in Experimental Vein Grafts
ACE
Hyperplasia
MARTIN K. O'DONOHOE, M.B., B.Sc., F.R.C.S.I.,* LEWIS B. SCHWARTZ, M.D.,* ZELKO S. RADIC, M.D.,* EILEEN M. MIKAT, PH.D.,t RICHARD L. McCANN, M.D.,* and PER-OTTO HAGEN, PH.D.*t
Intimal hyperplasia is an important factor in the pathophysiology of vein graft failure. Local renin-angiotensin systems recently have been shown to modulate the development of intimal hyperplasia in arteries after intimal injury. The effect of chronic angiotensin-converting enzyme (ACE) inhibition on the development of intimal hyperplasia in experimental vein grafts was examined in this study. Ten New Zealand White rabbits received 10 mg/kg of captopril daily in their drinking water. One week later the right carotid artery was divided and bypassed with the reversed right external jugular vein in these rabbits and in 10 matched controls. Captopril was continued for 28 days after operation, when all the grafts were harvested. Five grafts from each group were perfusion fixed, and the intimal thickness in the proximal, middle, and distal segments was determined. Rings from the remaining grafts (n = 20 in each group) were studied in vitro under isometric tension, and their responses to norepinephrine (NE), histamine (HIST), serotonin (5-HT), angiotensin I (Al), and angiotensin II (All) was measured. The intimal thickness of the proximal, middle, and distal segments of the captopril-treated grafts were significantly less than controls, being reduced in all segments by approximately 40% (p < 0.0001). With regard to vasoreactivity, the captopril-treated grafts were hypersensitive to 5-HT (control ED50 5.5 ± 0.5 X 10-7 mol/L vs. captopril-treated 1.1 ± 0.2 X 106 mol/L; p < 0.005) although the maximal response was significantly reduced (control 1.6 ± 0.3 g vs. captopril-treated 0.8 ± 0.1 g; p < 0.05). There were no differences in sensitivity between control and captopril-treated rings with respect to NE, HIST, AI, or All. Four of the ten captopril-treated segments, however, failed to respond to Al, and the maximal active tension of the responders was significantly reduced (control 0.47 ± 0.06 g vs. 0.20 ± 0.05 g; p < 0.02). These results suggest that ACE is involved in the modulation of vein graft intimal hyperplasia, and that ACE inhibitors may have therapeutic applications in patients undergoing vein bypass procedures. Presented at the Forum for Fundamental Problems in Surgery, American College of Surgeons Annual Clinical Congress, San Francisco, California, October 1990. Supported by U.S. Public Health Service Grants Nos. HL 15448, HL32720, and HL08086. Address reprint requests to Dr. Per-Otto Hagen, Atherosclerosis Research Laboratory, P.O. Box 3473, Duke University Medical Center, Durham, NC 27710. Accepted for publication May 22, 1991.
From the Departments of Surgery, * Pathology, t and Biochemistry,4 Duke University Medical Center, Durham, North Carolina
A UTOGENOUS SAPHENOUS VEIN is the preferred
conduit for most arterial bypass procedures where the vessel sizes are compatible and no suitable arterial graft is available. Vein graft failure, however, remains a significant clinical problem.'-3 The pathophysiology of vein graft failure has not been clearly elucidated. Autogenous vein grafts undergo structural and functional alterations after exposure to the arterial circulation. Intimal thickening develops in all veins within a short period after grafting. The pathologic significance of intimal thickening is uncertain, although some authors suggest that it may be a precursor to the development of atheroma and ultimate graft failure.`6 Localized regions ofintimal hyperplasia have been associated with graft stenoses and may be harbingers of graft occlusion.7 The role of the renin-angiotensin system in the pathogenesis of atherosclerosis is unclear. There is, however, convincing evidence that local renin-angiotensin systems exist in many arteries and veins.8'9 The activity of these systems results in the local production of angiotensin II independent of the systemic renin-angiotensin system. Angiotensin-converting enzyme (ACE) is a membranebound enzyme present in the endothelium of many large and medium-sized vessels in the peripheral circulation. It converts the relatively inactive angiotensin I (AI) to the potent vasoconstrictor angiotensin II (All). The smooth muscle cells of these vessels have numerous All receptors. All has been shown to stimulate hypertrophy in cultured smooth muscle cells and to induce production of the proto-oncogenes c-fos and c-myc, as well as in the A chain of platelet-derived growth factor (PDGF).1012 Local renin-angiotensin systems may be involved in
727
728
O'DONOHOE AND OTHERS
the response of blood vessels to intimal injury occurring after angioplasty, endarterectomy, and vein grafting. Inhibition of ACE has been shown to significantly reduce intimal hyperplasia after balloon-catheter injury in rat carotid artery and to have antiatherogenic effects in heritable hyperlipidemic rabbits and cholesterol-fed monkeys.'3-15 The aims of the present study were to examine the effect ofchronic ACE inhibition on the development of intimal hyperplasia and the vasomotor function of experimental vein grafts.
Materials and Methods Experimental Design Two groups of age and weight-matched adult male New Zealand White rabbits weighing 2 to 2.5 kg were studied. In 10 animals, captopril was administered in a dose of 10 mg/kg/day in the animals' drinking water; 10 additional animals were used as controls. One week later all animals underwent reversed autogenous vein bypass grafting of the right common carotid artery using the right external jugular vein. Blood pressure was measured at intervals during the study and the grafts were harvested after 28 days. Five grafts in each group were perfusion fixed for examination by light microscopy and the remainder studied in vitro under isometric tension. The grafts' responses to norepinephrine (NE), histamine (HIST), angiotensin I (Al), angiotensin II (AII), and serotonin (5-HT) were assessed. The animals received a normal diet during the period of study. Animal care complied with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1985).
Animal Operations General anesthesia was induced and maintained for all operations using ketamine (60 mg/kg), acepromazine (1 mg/kg), and xylazine (5 mg/kg) intramuscularly. A single dose of prophylactic antibiotic in the form of 30,000 IU/ kg of benzanthine and procaine penicillin (Durapen; Vedco, Inc., Overland Park, KS) was given intramuscularly at the time of induction. The right external jugular vein was harvested through a longitudinal neck incision as previously described.'6 The vein was stored in heparinized saline (5 IU/ml) while the common carotid artery was mobilized. The rabbit then was systemically heparinized (200 IU/kg IV), and a 4-cm section of the common carotid artery isolated between clamps. The vein was reversed and an end-to-side anastomosis was created with continuous 10-0 monofilament nylon suture (Ethicon, Inc., Somerville, NJ). The artery was ligated and divided between the anastomoses, leaving a graft of approximately 3 cm in length.
Ann. Surg. * December 1991
Four weeks later the animals were reanesthetized as described previously. The vein grafts from five animals in each group were left in situ and, after death, were fixed by perfusion and processed for histology. The vein grafts for in vitro study were removed atraumatically through longitudinal incisions and placed in oxygenated Krebs solution. Four rings, 4 to 5 mm in length, were cut from the central portion of each graft in preparation for isometric tension studies.
Blood Pressure Measurement The arterial pressure of all animals was measured through a cannula placed in the middle ear artery using a catheter-tipped pressure transducer (Millar Instruments Inc., Houston, TX). Measurements were obtained before entry into the study, at the time of vein grafting, and at the time of harvest. Blood pressure was continuously monitored with the animal awake in a quiet environment and the pressure recorded when stable. Histology The grafts chosen randomly for histology were exposed under general anesthesia. The animal was systemically heparinized (500 IU/kg) and killed with an overdose of sodium pentobarbital. The thoracic aorta was exposed and cannulated and the graft perfused at the animal's last recorded blood pressure with normal saline followed by 10% formaldehyde. After 30 minutes' fixation in situ, the grafts were removed, fixed overnight by immersion, and divided into proximal, middle, and distal segments. The segments were paraffin sectioned at 5 ,um and stained with modified Masson's trichrome and Verhoeffis elastic tissue stains.'7 The sections were examined by light microscopy in a blinded fashion. The intimal thickness was measured at 20 random points at even intervals around the circumference of each section using computerized videometric analysis (Innovision 150; American Innovision Inc., San Diego, CA); mean thickness for each graft segment was calculated from two separate sections. In Vitro Isometric Tension Studies
Vein graft rings from control and captopril-treated animals were suspended from stainless steel hooks in an organ bath containing oxygenated Krebs solution of the following composition: 122 mmol/L NaCl, 4.7 mmol/L KC1, 1.2 mmol/L MgCl2, 2.5 mmol/L CaCl2, 15.4 mmol/ L NaHCO3, 1.2 mmol/L KH2PO4, and 5.5 mmol/L glucose. The solution was maintained at 37 C and bubbled with a mixture of 95% 02 and 5% CO2. One hook was fixed to the bottom of the bath and the other connected to a force transducer (Myograph F-60; Narco Bio-Systems, Houston, TX). Responses were recorded on a multi-
Vol. 214 - No. 6
729
CAPTOPRIL REDUCES INTIMAL HYPERPLASIA
channel polygraph (Physiograph MK-l 1 l-S; Narco BioSystems). The optimal resting tension for each ring was determined by its maximal response to a modified oxygenated Krebs solution containing 60 mmol/L KCI, 66.7 mmol/L NaCl, 1.2 mmol/L MgCl2, 2.5 mmol/L CaCl2, 15.4 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, and 5.5 mmol/L glucose at resting tensions ranging from 0.4 to 2.5 g. The optimal resting isometric tension was applied and the tissue allowed to equilibrate in physiologic Krebs solution for 1 hour. The remainder of the experiments were performed at each ring's specific optimal resting tension. Norepinephrine (NE; Sigma Chemical Co., St. Louis, MO), dissolved in l0-3 mol/L HCI, was added in a cumulative manner to each organ bath chamber and the responses were recorded. After washout and re-equilibration for 45 minutes, dose-response curves were similarly obtained in response to histamine (HIST), angiotensin I (AI), angiotensin II (AII), and serotonin (5-HT; all from Sigma; all dissolved in distilled H20). To avoid tachyphylaxis to either AI or All, half the segments in each group were tested with only one of these agonists.
Statistics The isometric responses of the rings were subsequently converted to percent maximal response to yield standard
A
dose-response curves. The dose-response relationship was analyzed using logistic analysis to facilitate median effective dose (ED50) calculation.'8 The maximal response of each ring was expressed as grams of isometric tension developed. All data are expressed as the mean ± standard error of the mean. Differences in means were tested for significance using two-tailed Student's t test for unpaired data with p values < 0.05 regarded as significant.
Results All animals survived the procedure and all grafts were patent at the time of harvesting. There was no significant difference in mean arterial pressure between the control and captopril-treated animals. The optimal resting tension for the control grafts (0.99 ± 0.06 g) was significantly greater than the optimal resting tension for the captopriltreated grafts (0.75 ± 0.01 g; p < 0.001). Intimal Thickness All grafts in both groups exhibited some degree of intimal hyperplasia consisting of smooth muscle proliferation, the formation of a neo-intima, and the deposition ofcollagen and elastin in both the intima and media (Fig. 1). Morphometrically determined intimal thickness of the
B
FIG. 1. A, Control vein graft showing the degree of intimal hyperplasia after 4 weeks. B, captopril-treated vein graf, with approximately 40% reduction in intimal thickness. Modified Masson's trichrome and Verhoeffs elastic tissue stains (IH, intimal hyperplasia; M, media; AD, adventitia). Original magnification X200.
,40-
Al
%W
9
.V. 40 1141i.rb-
I
-
11%. mm:
00
1
Amlw.-.!e.w
dopt, lv. ;P.. .N
'Ago w
%AW.04.
A
-it -do A&
730
O'DONOHOE AND OTHERS
TABLE 1. Effect of Captopril on Vein Graft Intimal Thickness
Control Group Segment
(Am)
Captopril-treated Group (,m)
p
Proximal Middle Distal
156 ± 5 150 ± 8 151 ± 7
90 ± 4 95 ± 5 88 ± 3
Inhibition Reduces Intimal in Experimental Vein Grafts
ACE
Hyperplasia
MARTIN K. O'DONOHOE, M.B., B.Sc., F.R.C.S.I.,* LEWIS B. SCHWARTZ, M.D.,* ZELKO S. RADIC, M.D.,* EILEEN M. MIKAT, PH.D.,t RICHARD L. McCANN, M.D.,* and PER-OTTO HAGEN, PH.D.*t
Intimal hyperplasia is an important factor in the pathophysiology of vein graft failure. Local renin-angiotensin systems recently have been shown to modulate the development of intimal hyperplasia in arteries after intimal injury. The effect of chronic angiotensin-converting enzyme (ACE) inhibition on the development of intimal hyperplasia in experimental vein grafts was examined in this study. Ten New Zealand White rabbits received 10 mg/kg of captopril daily in their drinking water. One week later the right carotid artery was divided and bypassed with the reversed right external jugular vein in these rabbits and in 10 matched controls. Captopril was continued for 28 days after operation, when all the grafts were harvested. Five grafts from each group were perfusion fixed, and the intimal thickness in the proximal, middle, and distal segments was determined. Rings from the remaining grafts (n = 20 in each group) were studied in vitro under isometric tension, and their responses to norepinephrine (NE), histamine (HIST), serotonin (5-HT), angiotensin I (Al), and angiotensin II (All) was measured. The intimal thickness of the proximal, middle, and distal segments of the captopril-treated grafts were significantly less than controls, being reduced in all segments by approximately 40% (p < 0.0001). With regard to vasoreactivity, the captopril-treated grafts were hypersensitive to 5-HT (control ED50 5.5 ± 0.5 X 10-7 mol/L vs. captopril-treated 1.1 ± 0.2 X 106 mol/L; p < 0.005) although the maximal response was significantly reduced (control 1.6 ± 0.3 g vs. captopril-treated 0.8 ± 0.1 g; p < 0.05). There were no differences in sensitivity between control and captopril-treated rings with respect to NE, HIST, AI, or All. Four of the ten captopril-treated segments, however, failed to respond to Al, and the maximal active tension of the responders was significantly reduced (control 0.47 ± 0.06 g vs. 0.20 ± 0.05 g; p < 0.02). These results suggest that ACE is involved in the modulation of vein graft intimal hyperplasia, and that ACE inhibitors may have therapeutic applications in patients undergoing vein bypass procedures. Presented at the Forum for Fundamental Problems in Surgery, American College of Surgeons Annual Clinical Congress, San Francisco, California, October 1990. Supported by U.S. Public Health Service Grants Nos. HL 15448, HL32720, and HL08086. Address reprint requests to Dr. Per-Otto Hagen, Atherosclerosis Research Laboratory, P.O. Box 3473, Duke University Medical Center, Durham, NC 27710. Accepted for publication May 22, 1991.
From the Departments of Surgery, * Pathology, t and Biochemistry,4 Duke University Medical Center, Durham, North Carolina
A UTOGENOUS SAPHENOUS VEIN is the preferred
conduit for most arterial bypass procedures where the vessel sizes are compatible and no suitable arterial graft is available. Vein graft failure, however, remains a significant clinical problem.'-3 The pathophysiology of vein graft failure has not been clearly elucidated. Autogenous vein grafts undergo structural and functional alterations after exposure to the arterial circulation. Intimal thickening develops in all veins within a short period after grafting. The pathologic significance of intimal thickening is uncertain, although some authors suggest that it may be a precursor to the development of atheroma and ultimate graft failure.`6 Localized regions ofintimal hyperplasia have been associated with graft stenoses and may be harbingers of graft occlusion.7 The role of the renin-angiotensin system in the pathogenesis of atherosclerosis is unclear. There is, however, convincing evidence that local renin-angiotensin systems exist in many arteries and veins.8'9 The activity of these systems results in the local production of angiotensin II independent of the systemic renin-angiotensin system. Angiotensin-converting enzyme (ACE) is a membranebound enzyme present in the endothelium of many large and medium-sized vessels in the peripheral circulation. It converts the relatively inactive angiotensin I (AI) to the potent vasoconstrictor angiotensin II (All). The smooth muscle cells of these vessels have numerous All receptors. All has been shown to stimulate hypertrophy in cultured smooth muscle cells and to induce production of the proto-oncogenes c-fos and c-myc, as well as in the A chain of platelet-derived growth factor (PDGF).1012 Local renin-angiotensin systems may be involved in
727
728
O'DONOHOE AND OTHERS
the response of blood vessels to intimal injury occurring after angioplasty, endarterectomy, and vein grafting. Inhibition of ACE has been shown to significantly reduce intimal hyperplasia after balloon-catheter injury in rat carotid artery and to have antiatherogenic effects in heritable hyperlipidemic rabbits and cholesterol-fed monkeys.'3-15 The aims of the present study were to examine the effect ofchronic ACE inhibition on the development of intimal hyperplasia and the vasomotor function of experimental vein grafts.
Materials and Methods Experimental Design Two groups of age and weight-matched adult male New Zealand White rabbits weighing 2 to 2.5 kg were studied. In 10 animals, captopril was administered in a dose of 10 mg/kg/day in the animals' drinking water; 10 additional animals were used as controls. One week later all animals underwent reversed autogenous vein bypass grafting of the right common carotid artery using the right external jugular vein. Blood pressure was measured at intervals during the study and the grafts were harvested after 28 days. Five grafts in each group were perfusion fixed for examination by light microscopy and the remainder studied in vitro under isometric tension. The grafts' responses to norepinephrine (NE), histamine (HIST), angiotensin I (Al), angiotensin II (AII), and serotonin (5-HT) were assessed. The animals received a normal diet during the period of study. Animal care complied with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1985).
Animal Operations General anesthesia was induced and maintained for all operations using ketamine (60 mg/kg), acepromazine (1 mg/kg), and xylazine (5 mg/kg) intramuscularly. A single dose of prophylactic antibiotic in the form of 30,000 IU/ kg of benzanthine and procaine penicillin (Durapen; Vedco, Inc., Overland Park, KS) was given intramuscularly at the time of induction. The right external jugular vein was harvested through a longitudinal neck incision as previously described.'6 The vein was stored in heparinized saline (5 IU/ml) while the common carotid artery was mobilized. The rabbit then was systemically heparinized (200 IU/kg IV), and a 4-cm section of the common carotid artery isolated between clamps. The vein was reversed and an end-to-side anastomosis was created with continuous 10-0 monofilament nylon suture (Ethicon, Inc., Somerville, NJ). The artery was ligated and divided between the anastomoses, leaving a graft of approximately 3 cm in length.
Ann. Surg. * December 1991
Four weeks later the animals were reanesthetized as described previously. The vein grafts from five animals in each group were left in situ and, after death, were fixed by perfusion and processed for histology. The vein grafts for in vitro study were removed atraumatically through longitudinal incisions and placed in oxygenated Krebs solution. Four rings, 4 to 5 mm in length, were cut from the central portion of each graft in preparation for isometric tension studies.
Blood Pressure Measurement The arterial pressure of all animals was measured through a cannula placed in the middle ear artery using a catheter-tipped pressure transducer (Millar Instruments Inc., Houston, TX). Measurements were obtained before entry into the study, at the time of vein grafting, and at the time of harvest. Blood pressure was continuously monitored with the animal awake in a quiet environment and the pressure recorded when stable. Histology The grafts chosen randomly for histology were exposed under general anesthesia. The animal was systemically heparinized (500 IU/kg) and killed with an overdose of sodium pentobarbital. The thoracic aorta was exposed and cannulated and the graft perfused at the animal's last recorded blood pressure with normal saline followed by 10% formaldehyde. After 30 minutes' fixation in situ, the grafts were removed, fixed overnight by immersion, and divided into proximal, middle, and distal segments. The segments were paraffin sectioned at 5 ,um and stained with modified Masson's trichrome and Verhoeffis elastic tissue stains.'7 The sections were examined by light microscopy in a blinded fashion. The intimal thickness was measured at 20 random points at even intervals around the circumference of each section using computerized videometric analysis (Innovision 150; American Innovision Inc., San Diego, CA); mean thickness for each graft segment was calculated from two separate sections. In Vitro Isometric Tension Studies
Vein graft rings from control and captopril-treated animals were suspended from stainless steel hooks in an organ bath containing oxygenated Krebs solution of the following composition: 122 mmol/L NaCl, 4.7 mmol/L KC1, 1.2 mmol/L MgCl2, 2.5 mmol/L CaCl2, 15.4 mmol/ L NaHCO3, 1.2 mmol/L KH2PO4, and 5.5 mmol/L glucose. The solution was maintained at 37 C and bubbled with a mixture of 95% 02 and 5% CO2. One hook was fixed to the bottom of the bath and the other connected to a force transducer (Myograph F-60; Narco Bio-Systems, Houston, TX). Responses were recorded on a multi-
Vol. 214 - No. 6
729
CAPTOPRIL REDUCES INTIMAL HYPERPLASIA
channel polygraph (Physiograph MK-l 1 l-S; Narco BioSystems). The optimal resting tension for each ring was determined by its maximal response to a modified oxygenated Krebs solution containing 60 mmol/L KCI, 66.7 mmol/L NaCl, 1.2 mmol/L MgCl2, 2.5 mmol/L CaCl2, 15.4 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, and 5.5 mmol/L glucose at resting tensions ranging from 0.4 to 2.5 g. The optimal resting isometric tension was applied and the tissue allowed to equilibrate in physiologic Krebs solution for 1 hour. The remainder of the experiments were performed at each ring's specific optimal resting tension. Norepinephrine (NE; Sigma Chemical Co., St. Louis, MO), dissolved in l0-3 mol/L HCI, was added in a cumulative manner to each organ bath chamber and the responses were recorded. After washout and re-equilibration for 45 minutes, dose-response curves were similarly obtained in response to histamine (HIST), angiotensin I (AI), angiotensin II (AII), and serotonin (5-HT; all from Sigma; all dissolved in distilled H20). To avoid tachyphylaxis to either AI or All, half the segments in each group were tested with only one of these agonists.
Statistics The isometric responses of the rings were subsequently converted to percent maximal response to yield standard
A
dose-response curves. The dose-response relationship was analyzed using logistic analysis to facilitate median effective dose (ED50) calculation.'8 The maximal response of each ring was expressed as grams of isometric tension developed. All data are expressed as the mean ± standard error of the mean. Differences in means were tested for significance using two-tailed Student's t test for unpaired data with p values < 0.05 regarded as significant.
Results All animals survived the procedure and all grafts were patent at the time of harvesting. There was no significant difference in mean arterial pressure between the control and captopril-treated animals. The optimal resting tension for the control grafts (0.99 ± 0.06 g) was significantly greater than the optimal resting tension for the captopriltreated grafts (0.75 ± 0.01 g; p < 0.001). Intimal Thickness All grafts in both groups exhibited some degree of intimal hyperplasia consisting of smooth muscle proliferation, the formation of a neo-intima, and the deposition ofcollagen and elastin in both the intima and media (Fig. 1). Morphometrically determined intimal thickness of the
B
FIG. 1. A, Control vein graft showing the degree of intimal hyperplasia after 4 weeks. B, captopril-treated vein graf, with approximately 40% reduction in intimal thickness. Modified Masson's trichrome and Verhoeffs elastic tissue stains (IH, intimal hyperplasia; M, media; AD, adventitia). Original magnification X200.
,40-
Al
%W
9
.V. 40 1141i.rb-
I
-
11%. mm:
00
1
Amlw.-.!e.w
dopt, lv. ;P.. .N
'Ago w
%AW.04.
A
-it -do A&
730
O'DONOHOE AND OTHERS
TABLE 1. Effect of Captopril on Vein Graft Intimal Thickness
Control Group Segment
(Am)
Captopril-treated Group (,m)
p
Proximal Middle Distal
156 ± 5 150 ± 8 151 ± 7
90 ± 4 95 ± 5 88 ± 3
